Mucosal immunization with live recombinant bovine respiratory syncytial virus (BRSV) and recombinant BRSV lacking the envelope glycoprotein G protects against challenge with wild-type BRSV

Abstract

Recombinant bovine respiratory syncytial virus (rBRSV) and an rBRSV deletion mutant lacking the G gene (rBRSVDeltaG) were characterized in calves with respect to replication competence, attenuation, and protective efficacy as live-attenuated BRSV vaccines. Both recombinant viruses were safe and induced protection against a BRSV challenge infection. rBRSV replicated efficiently in the upper respiratory tract. Intranasal immunization with rBRSVDeltaG led to infection but not to mucosal virus replication. Neutralizing antibodies were induced by rBRSV and rBRSVDeltaG. Thus, the BRSV attachment glycoprotein G seems to be dispensable in vaccinating calves against BRSV.

FIG. 1.

Detection of rBRSVΔG mRNA transcription. To detect BRSV mRNA transcription in the rBRSVΔG immunized group, RNA was prepared from nasal swab material and RT-PCR was performed. First-strand cDNA was synthezised using a primer complementary to the N mRNA (ATue51908; nt 1676 to 1657), and RT-PCR was done using a primer downstream of the first-strand primer (ATue51908; nt 1613 to 1594) and a primer with the sequence of the N open reading frame 3′ end (ATue51908; nt 1144 to 1163), resulting in an RT-PCR product of 470 bp. +, positive control (RT-PCR from total RNA from rBRSVΔG-infected cells); -, negative control.

FIG. 2.

Differentiation of rBRSV and wild-type BRSV isolated from nasal swabs using a synthetic marker restriction site. Top, RT-PCR was performed with total RNA of cell cultures inoculated with nasal swab material (RNeasy; Qiagen). RNAs from recombinant viruses or from strain CA-1 were reverse transcribed using a positive-sense primer which was complementary to the BRSV F gene (ATue51908; nt 7218 to 7240). An aliquot of the first-strand cDNA was used for PCR with the first-strand primer and a BRSV M2-specific primer (ATue51908; nt 7852 to 7832, negative sense). RT-PCR products were analyzed on 3% agarose gels. The RT-PCR products were consistent with the predicted sizes. Digestion with XhoI yielded the expected fragments for rBRSV. As expected, the RT-PCR products resulting from the challenge BRSV, strain CA-1, remained uncleaved. The bottom panel gives a schematic overview of the locations of primers and XhoI marker restriction site, with the sizes of the resulting fragments indicated. control, RT-PCR performed on total RNA of cells infected with rBRSV.